Beschreibung:
Abstract Lung cancer, especially adenocarcinoma as the most common subtype, is still the most deadly human cancers. Therefore biomarker discovery for implementation of individualised therapies remains of utmost importance. Serine hydrolases (SHs), one of the largest enzyme families, have previously been shown to be implicated in the development of lung cancers. Activity-based protein profiling (ABPP) is a proteomic method that uses active site-directed chemical probes to selectively target the active form of the subsets of the enzymes in question, and then by a combination of a streptavidin-biotin enrichment step and mass spectrometry quantifies the catalytically active amount of the enzyme molecule. In this project, we monitored both forms of serine hydrolase, the catalytically active and inactive, in a patient cohort of lung adenocarcinoma biopsies. For this purpose, we combined the activity-based proteomics for serine hydrolase and SWATH mass spectrometry (MS), which ensures highly reproducible protein quantification in a large panel of clinical samples. Twenty four lung biopsies of long- and short surviving patients with stage IIIA adenocarcinomas and their normal tissue counterparts were available as OCT-embedded tissue. Although OCT represents a non-reactive compound used to preserve tissue morphology in long-term sample storage, this substance is incompatible with mass spectrometry measurements. We optimized and developed an OCT clean-up protocol compatible with enzyme-substrate binding of activity-based chemical probes and the targeted portion of the active enzyme. To prevent digestion on streptavidin beads and MS spectra contamination, we used the reproducible and accurate SWATH MS to indirectly measure the active enzyme form in the biopsy-extract solution which had earlier been depleted for all active enzyme molecules by precipitation with chemical probes attached to the beads. We identified over 4000 lung tissue proteins from a few milligrams of OCT-embedded biopsies, and confirmed good data quality for further SH enzyme quantification. In addition to the analysis of total proteome, 278 distinct proteins were identified on the parallel streptavidin bead samples, with chemical probes being used to deplete the active enzyme from the biopsy-extract. Each SWATH experiment reported the percentage of active enzyme form through the indirectly measured ratio between the inactive SHs (sample depleted for active SHs) and the total SHs (non-depleted sample). We detected around 80 enzymes with the active form comparably measured in all three independent experiments, which amount generally accounted for between 5-55% of the total enzyme concentration. We conclude that with the modified protocol compatible with OCT-embedded tissue biopsies the combination of ABPP and SWATH-MS enables highly reproducible protein quantification in biomarker discovery. Citation Format: Tatjana Sajic, Stephan Arni, Walter Weder, Rudolf Aebersold, Sven Hillinger. Modified SWATH MS analysis of serine hydrolase activity in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4934.